plants
Article
In Vitro Root Induction from Argan (Argania spinosa (L.)
Skeels) Adventitious Shoots: Influence of Ammonium Nitrate,
Auxins, Silver Nitrate and Putrescine, and Evaluation of
Plantlet Acclimatization
Ilham Amghar 1,2 , Mohammed Ibriz 2 , Maha Ibrahimi 1 , Abdelaali Boudra 3 , Fatima Gaboun 1 , Reda Meziani 4 ,
Driss Iraqi 1 , Mouaad Amine Mazri 5 , Ghizlane Diria 1 and Rabha Abdelwahd 1, *






Citation: Amghar, I.; Ibriz, M.;
Ibrahimi, M.; Boudra, A.; Gaboun, F.;
Meziani, R.; Iraqi, D.; Mazri, M.A.;
Diria, G.; Abdelwahd, R. In Vitro
Root Induction from Argan (Argania
spinosa (L.) Skeels) Adventitious
Shoots: Influence of Ammonium
Nitrate, Auxins, Silver Nitrate and
Putrescine, and Evaluation of Plantlet
Acclimatization. Plants 2021, 10, 1062.
https://doi.org/10.3390/
plants10061062
Academic Editors:
Hironaka Tsukagoshi,
Takehiro Kamiya and
Mary Paz González García
Received: 3 March 2021
Accepted: 29 March 2021
Published: 26 May 2021
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Copyright: © 2021 by the authors.
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This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Plants 2021, 10, 1062. https://doi.org/10.3390/plants10061062
1 UR Biotechnologie, CRRA-Rabat, Institut National de la Recherche Agronomique, BP 6570,
Rabat 10101, Morocco; amghar.ilham@gmail.com (I.A.); maha.ibrahimi@gmail.com (M.I.);
gabounf@gmail.com (F.G.); iraqid@yahoo.fr (D.I.); ghizlanediria@gmail.com (G.D.)
2 Laboratoire de Génétique et Biométrie, Département de Biologie, Faculté des Sciences de Kénitra,
Université Ibn Tofail, BP 133, Kenitra 14000, Morocco; m_ibriz@yahoo.fr
3 Office National du Conseil Agricole, Agadir 80000, Morocco; boudra_1999@yahoo.fr
4 Laboratoire National de Culture des Tissus du Palmier Dattier, UR Systèmes Oasiens, CRRA-Errachidia,
Institut National de la Recherche Agronomique, Avenue Moulay Ali Cherif, BP 2, Errachidia 52000, Morocco;
redameziani@yahoo.fr
5 Laboratoire de Biotechnologie Végétale, UR Agro-Biotechnologie, CRRA-Marrakech, Institut National de la
Recherche Agronomique, BP 533, Marrakech 40000, Morocco; m.a.mazri@gmail.com
* Correspondence: rabhaab@yahoo.fr or rabha.abdelwahd@inra.ma
Abstract: Argania spinosa (L.) Skeels is an endangered plant species endemic to Morocco. In recent
years, attempts to develop in vitro regeneration systems for this species were made. However,
rooting and acclimatization of in vitro plants have been a bottleneck for successful propagation. In
the present study, the effects of different concentrations of auxins, putrescine, silver nitrate (AgNO3 )
and ammonium nitrate on the in vitro rooting of adventitious shoots of two argan genotypes “Mejji”
and “R’zwa”, were evaluated. The highest rooting percentages (86.6% in “Mejji” and 84.4% in
“R’zwa”) were observed on Murashige and Skoog (MS) medium modified by reducing the ammonium
nitrate concentration and supplemented with 1.5 mg L−1 indole-3-butyric acid (IBA), 0.5 mg L−1 1-
naphthalene acetic acid (NAA), 2 mg L−1 AgNO3 and 160 mg L−1 putrescine. This medium resulted
in the development of a good root system after only 10 days of culture. Plantlet acclimatization was
carried out using different substrate mixtures, and high survival rates (100%) were observed when
the substrate contained either peat alone or a sand–peat mixture (1:1, w/w). The high percentages
of rooting and acclimatization reported in the present study are of high importance for rapid and
large-scale propagation of this endangered species.
Keywords: micropropagation; organogenesis; plant regeneration; rhizogenesis
1. Introduction
Argan (Argania spinosa (L.) Skeels) is a forest species belonging to the family Sapotaceae
growing endemically in Morocco [1,2]. Argan plays multiple socioeconomic and envi-
ronmental roles. In fact, argan fruits are used by local populations for the production of
argan oil, a product that has long been valued for its nutritional, medicinal and therapeu-
tic properties [3,4]. Argan oil is one of the most expensive and sought-after oils in the
world. The price of argan oil in the international market exceeds US$400 per liter [5]. The
argan oil industry significantly contributes to the income of local populations and sustains
and improves their livelihood [6]. On the other hand, the argan ecosystem was reported
to improve water quality, crop production and rangeland conditions and contributes to
maintaining high soil quality, protecting biodiversity and controlling desertification [7].
https://www.mdpi.com/journal/plants
Plants 2021, 10, 1062 2 of 15

Despite the high socioeconomic and ecological importance of argan, this species is
threatened by several biotic and abiotic factors that led to the argan ecosystem’s degrada-
tion. For example, argan is overexploited by local people for food, wood, cosmetic and
medicine industries [8]. In addition, argan suffers from intensive overgrazing by goats and
natural habitat loss by urban expansion [8–10]. Developing efficient propagation systems
for argan is today a key tool in the preservation and rehabilitation of the argan ecosystem.
The natural propagation of argan by seed germination and seedling growth is con-
strained by a wide range of factors. The most important among them is that argan is a
slow-growing species; thus, seedlings hardly survive harsh environmental conditions and
are permanently exposed to the risk of goat overgrazing [9,11]. Vegetative propagation
through stem cuttings cannot be envisaged for the preservation and large-scale multipli-
cation because of the recalcitrance of argan plants to rooting [12,13]. In vivo grafting is
another approach used for the propagation of superior argan genotypes. The success of this
technique depends on the environmental conditions, the degree of compatibility between
the rootstock and scion and the physiological activity of scions [14].
Plant cell and tissue culture is a powerful tool for rapid and large-scale propagation of
endangered plant species [15,16]. In the case of argan, this technology has not been well
explored even though some studies were recently published [13,17–19]. Koufan et al. [17]
and Lamaoui et al. [19] evaluated the effects of different plant growth regulators (PGRs) on
bud break, shoot multiplication, elongation and adventitious root induction from argan
microcuttings and concluded that the application of this technique is hindered by the
difficulty to induce root formation from microcuttings. Koufan et al. [13] have described
a novel approach for argan micropropagation by using in vitro grafting. This technique
allows overcoming the rooting problems of microcuttings. However, plantlet survival
during acclimatization should be improved [13]. Developing efficient regeneration systems
through somatic embryogenesis or organogenesis will undoubtedly contribute to the
propagation, genetic improvement and preservation of argan, as was the case in other
oil crops [20–23]. Recently, a regeneration system has been established for argan through
organogenesis [24]. The development of an efficient and reproducible organogenesis
system strongly depends on successful in vitro rooting of the regenerated shoots and high
survival rates during acclimatization to ex vitro conditions.
Rooting is a crucial step in the micropropagation of plant species [25]. The root
system plays important roles in water and nutrient uptakes, gas transport processes,
plant growth and development and in the defense mechanisms against biotic and abiotic
stresses [26,27]. The in vitro rooting process is governed by diverse genetic factors and is
greatly influenced by culture medium components, mainly the type and concentration of
PGRs and polyamines [28–30]. On the other hand, plantlet survival in ex vitro conditions is
significantly affected by the planting substrate, which is one of the main factors determining
plant growth and development [31,32]. In argan, different potting substrates were used
for the acclimatization of micropropagated plants. Nouaim et al. [12] planted argan
microcuttings in a substrate composed of Terragreen saturated with “Long Ashton” nutrient
solution. Koufan et al. [13] and Lamaoui et al. [19] used a mixture of peat and sand, while
Justamante et al. [18] used a peat–perlite mix.
The present study aimed to determine the effects of nitrate, putrescine, and PGRs
on in vitro root induction and growth from adventitious shoots of two argan genotypes,
“Mejji” and “R’zwa”, and to select the best planting substrate for improved plant survival
under ex vitro conditions.

2. Results
2.1. Effects of Putrescine, Silver Nitrate (AgNO3 ), Ammonium Nitrate (NH4 NO3 ),
Indole-3-Butyric Acid (IBA) and 1-Naphthalene Acetic Acid (NAA) on In Vitro Rooting of
Regenerated Shoots
2.1.1. Effects of NH4 NO3 on In Vitro Rooting
The concentration of NH4 NO3 had a significant effect on argan shoot rooting (Table 1).
Indeed, when the culture media were supplemented with both IBA and putrescine,
 Plants 2021, 10, 1062 3 of 15

Murashige and Skoog (MS) medium showed significantly higher rooting percentages
(52.2% in “Mejji” and 57.7% in “R’zwa”) than RM (MS modified by reducing NH4 NO3
concentration to 825 mg L−1 ) and M (MS without NH4 NO3 ) media. However, the shoots
cultured on RM medium showed the highest average number of roots after 75 days of
culture (4.6 in “Mejji” and 4.4 in “R’zwa”). In the M medium, significantly lower rooting
percentages were observed (Table 1).

2.1.2. Effects of IBA, AgNO3 and Putrescine on In Vitro Rooting

In both argan genotypes, no root formation was observed in auxin-free media. Like-
wise, the use of either putrescine, AgNO3 or IBA alone did not induce adventitious rooting
in micropropagated shoots. Rooting occurred in shoots cultured on media containing
at least one auxin (IBA or NAA) plus putrescine and/or AgNO3 (Table 1). The shoots
0, x FOR PEER REVIEW cultured on MS medium supplemented with 1.5 mg L−1 IBA and 160 mg L−1 putrescine
showed rooting percentages of 57.7% and 52.2% in “R’zwa” and “Mejji”, respectively. In
all putrescine-IBA-containing media, root induction required 8 weeks, while 2 more weeks
were needed for root development (Figure 1). In some cases, callus formation was observed
re means ± SD. Values in the same column and with different letter(s) are significantly different at p < 0.05. ǂ indicates significance between genotypes
at the
, Treatment. MS, Murashige and Skoog basal cut end
medium; RM,ofMurashige
shoots. Thisand results in low-quality
Skoog basal medium, but roots, not 825
contains suitable
mg L−1for
NHacclimatization
4NO3; M: Murashige and
(Table 1 and Figure 2).
medium without NH4NO3. Callus formation: 0C, no callus; C+, low callus formation; C++, moderate callus formation; C+++, high callus formation.

e 1. In vitro rooting kinetic

Figure 1. Inofvitro
the two genotypes
rooting kinetic of
of Argania Spinosa in different
the two genotypes of Arganiaculture media
Spinosa mentioned
in different in Table
culture media1.mentioned
(a) genotype “Mejji”;
in Table (b) genotype “R’
1. (a)
genotype “Mejji”; (b) genotype “R’zwa”.
Plants 2021, 10, 1062 4 of 15

Table 1. Effect of genotype, putrescine, silver nitrate, indole-3-butyric acid (IBA) and 1-naphthalene acetic acid (NAA) on rooting percentage, number of roots per shoot, root length, callus
formation and the time required for rooting in two argan (Argania spinosa (L.) Skeels) genotypes, “Mejji” and “R’zwa”.

PGRs (mg
“Mejji” “R’zwa”
L−1 )
Basal AgNO3 Putrescine Average
Treatment (mg L−1 ) Rooting Average No. Rooting Average No. Average
Medium (mg L−1 ) Root Callus Time for
Callus
Time for
IBA NAA Frequency of Frequency of Root Length
(%) Roots/Shoot Length Formation Rooting
(Days) (%) Roots/Shoot (cm) Formation Rooting
(Days)
(cm)
T0 MS - - - - 0 0 0 0C - 0 0 0 0C -
T1 MS - - 1.5 - 0 0 0 0C - 0 0 0 0C -
T2 MS - 160 - - 0 0 0 0C - 0 0 0 0C -
T3-T4-T5 MS 2002/4/6 - - - 0 0 0 0C - 0 0 0 0C -
T6 MS - 160 1.5 - 52.2 ± 1.1 c 3.1 ± 0.2 d,e 2.8 ± 0.2 a C+++ 90 57.7 ± 2.9 c 2.8 ± 0.2 a 3.4 ± 0.2 a,b,c C+++ 90
T7 RM - 160 1.5 - 43.3 ± 3.8 d 4.6 ± 0.2 c 3.1 ± 0.2 a C++ 75 47.7 ± 2.9 d 4.4 ± 0.2 c 2.8 ± 0.2 a,b,c C+++ 75
T8 M - 160 1.5 - 19.9 ± 1.9 f,g 2.5 ± 0.3 e,f,g 2.0 ± 0.1 b,c C++ 60 21.1 ± 2.2 f,g 2.8 ± 0.3 e,f 3.0 ± 0.1 a,b,c C+++ 60
T9 MS 2 - 1.5 - 39.9 ± 1.9 d 3.3 ± 0.2 d,e 3.3 ± 0.3 a 0C 30 43.3 ± 1.9 de 3.1 ± 0.2 d,e,f 2.7 ± 0.2 a,b,c 0C 30
T10 MS 4 - 1.5 - 32.2 ± 1.1 d,e 1.9 ± 0.1 f,g 1.6 ± 0.1 c 0C 30 38.8 ± 1.1 e 2.2 ± 0.1 f,g 2.3 ± 0.1 c 0C 30
T11 MS 6 - 1.5 - 19.9 ± 1.9 f,g 1.9 ± 0.2 f,g 2.8 ± 0.4 a,b 0C 30 18.8 ± 2.9 g 1.5 ± 0.1 g 2.4 ± 0.3 b,c 0C 30
T12 MS 2 160 1.5 - 61.1 ± 2.9 b 3.2 ± 0.1 d,e 3.3 ± 0.2 a 0C 20 63.3 ± 3.3 b,c 3.3 ± 0.1 c,d 3.2 ± 0.1 a,b 0C 20
T13 RM 2 160 1.5 - 65.5 ± 4.8 b 3.7 ± 0.2 c,d 3.2 ± 0.2 a 0C 20 67.7 ± 4.8 c 3.8 ± 0.2 c,d 3.3 ± 0.2 a 0C 20
T14 RM 2 160 1.5 0.5 86.6 ± 3.8 a 6.3 ± 0.3 b 3.5 ± 0.1 a 0C 10 84.4 ± 2.9 a 6.7 ± 0.3 b 3.4 ± 0.1 a C+ 10
T15 RM 2 160 1.5 1 38.8 ± 1.1 c,d 2.9 ± 0.1 d,e,f 3.2 ± 0.2 a C+ 20 38.8 ± 1.1 e 3.3 ± 0.2 d,e 2.9 ± 0.2 a,b,c C++ 20
T16 RM 2 160 1.5 1.5 16.6 ± 1.9 g 1.6 ± 0.1 g 1.4 ± 0.2 c C+++ 25–30 17.7 ± 2.9 g 2.6 ± 0.3 s,f 1.4 ± 0.1 d C+++ 25–30
T17 RM 2 160 - 1.5 26.6 ± 1.9 e,f 10.1 ± 0.6 a 1.6 ± 0.1 c C+++ 25 27.7 ± 1.1 f 10.5 ± 0.4 a 1.5 ± 0.1 d C+++ 25
Data are means ± SD. Values in the same column and with different letter(s) are significantly different at p < 0.05. T, Treatment. MS, Murashige and Skoog basal medium; RM, Murashige and Skoog basal
medium, but contains 825 mg L−1 NH4 NO3 ; M: Murashige and Skoog basal medium without NH4 NO3 . Callus formation: 0C, no callus; C+, low callus formation; C++, moderate callus formation; C+++, high
callus formation.
Plants 2021, 10, 1062 5 of 15
Plants 2021, 10, x FOR PEER REVIEW 6 of 16

Figure
Figure 2. In2.vitro
In vitro rooting
rooting of different
of different arganargan genotypes.
genotypes. (a) In(a)vitro
In vitro rooting
rooting in the
in the presence
presence of putrescine
of putrescine andand indole-3-
indole-3-butyric
butyric acid (IBA) in Murashige and Skoog (MS) medium; (b)
acid (IBA) in Murashige and Skoog (MS) medium; (b) in vitro rooting in the presence in vitro rooting in the presence
of putrescine and IBA in MSinmedium
of putrescine and IBA MS
medium modified by reducing ammonium nitrate concentration to−825 1 mg L−1 (RM medium); (c) in vitro rooting in the
modified by reducing ammonium nitrate concentration to 825 mg L (RM medium); (c) in vitro rooting in the presence of
presence of putrescine and IBA in MS medium without ammonium nitrate (M medium); (d) in vitro rooting in the presence
putrescine
of silverand IBA(AgNO
nitrate in MS 3medium
) (2 mg L−1without
) and IBA; ammonium
(e) in vitro nitrate
rooting (M medium);
in the presence(d) in vitro3 (4
of AgNO rooting
mg L−1in) andtheIBA;
presence of silver
(f) in vitro
nitrate (AgNO − 1 − 1
rooting 3 ) (2
in the mg L of
presence ) and
AgNO IBA;
3 (6(e)
mginLvitro) androoting
IBA; (g)ininthe presence
vitro rootingofinAgNO 3 (4 mgofLAgNO
the presence ) and
3 (2IBA;
mg L (f)),inputrescine
vitro rooting
−1 −1

and
in the IBA; (h)ofinAgNO
presence vitro rooting
3 (6 mg in L−RM
1 ) and
medium
IBA; (g)containing
in vitro AgNO
rooting 3 (2inmg −1), putrescine and IBA; (i) in−vitro
theLpresence of AgNO3 (2 mg L 1 ), putrescine rooting inand RMIBA;
medium containing AgNO 3 (2 mg L−1), putrescine, IBA and 1-naphthalene− 1 acetic acid
(h) in vitro rooting in RM medium containing AgNO3 (2 mg L ), putrescine and IBA; (i) in vitro rooting in RM medium(NAA) (0.5 mg L −1); (j) in vitro root-

ing in RM medium containing AgNO3 (2 mg L−1), putrescine, IBA and NAA (1 mg L−1); (k) in vitro rooting in RM medium
containing AgNO3 (2 mg L−1 ), putrescine, IBA and 1-naphthalene acetic acid (NAA) (0.5 mg L−1 ); (j) in vitro rooting in
RM medium containing AgNO3 (2 mg L−1 ), putrescine, IBA and NAA (1 mg L−1 ); (k) in vitro rooting in RM medium
containing AgNO3 (2 mg L−1 ), putrescine, IBA and NAA (1.5 mg L−1 ); (l) in vitro rooting in RM medium containing
AgNO3 (2 mg L−1 ), putrescine and NAA (1.5 mg L−1 ). (1 ) indicates genotype “Mejji”, and (2 ) indicates genotype “R’zwa”.
Plants 2021, 10, 1062 6 of 15

In MS medium, the combination of AgNO3 and IBA resulted in rooting percentages

ranging from 19.9 to 39.9% in genotype “Mejji”, and from 18.8 to 43.3% in genotype
“R’zwa”, depending on AgNO3 concentration. In fact, AgNO3 concentration significantly
affected root induction, as well as the average number of roots per shoot and root length
(Table 1). Increasing the concentration of AgNO3 to 4 and 6 mg L−1 decreased the rooting
percentage and the average number of roots per shoot (Table 1).

2.1.3. Evaluation of the Combined Effects of Putrescine, AgNO3 , IBA and NAA on
In Vitro Rooting
To improve in vitro rooting and the quality of the root system, the combined effect
of NH4 NO3 , putrescine, AgNO3 , IBA and NAA was evaluated (Figure 2). The results
showed that combining IBA, putrescine and 2 mg L−1 AgNO3 significantly reduced the
root induction time to 20 days in MS and RM media in both argan genotypes. Adding
NAA at a low concentration (0.5 mg L−1 ) resulted in the highest rooting percentages
(86.6% in “Mejji” and 84.4% in “R’zwa”), an average root length of 3.5 cm in “Mejji” and
3.4 cm in “R’zwa”, and an average number of roots per shoot of 6.3 in “Mejji” and 6.7
in “R’zwa”. This combination showed root emergence within the first 10 days of culture
(Figure 1). The average number of roots per shoot depended on NAA concentration, with
the highest number of roots per shoot (10.1 in “Mejji” and 10.5 in “R’zwa”) observed when
1.5 mg L−1 NAA was added (Table 1). However, the use of 1.5 mg L−1 NAA resulted in
high callogenesis and low root quality.

2.1.4. Effects of Putrescine, AgNO3 , IBA and NAA on Rooting Kinetics

The results of the present study showed that culture medium components have a
significant impact on root induction. Shoot rooting started after 55 to 85 days of culture
in media supplemented with putrescine, whereas, in media supplemented with AgNO3 ,
rooting started after 25 days of culture. In both genotypes, when the shoots were cultured
on RM medium supplemented with 160 mg L−1 putrescine, 2 mg L−1 AgNO3 , 1.5 mg L−1
IBA and 0.5 mg L−1 NAA, rooting was observed after only 5 days of culture (Figure 1).
Moreover, the rooting percentage reached 50% and 80% in genotype “Mejji” after 15 and
20 days of culture, respectively.

2.2. Effect of Different Substrate Mixtures on Ex Vitro Acclimatization

The substrate significantly influenced the survival rate of the regenerated plantlets
(Table 2). The highest survival rate (100%) was observed when the plantlets of both
genotypes were transplanted into peat substrate or in the peat–sand mixture. When the
plantlets were transplanted into a sand substrate, the survival rate differed between the
two genotypes (Table 2). In fact, a survival rate of 100% was observed in “Mejji” plantlets,
while the plantlets of “R’zwa” showed a survival rate of 86.6%. The substrate mixture
composed of peat, sand, and forest soil showed lower survival rates (73.3 and 83.3% in
“Mejji” and “R’zwa”, respectively).
The substrate also influenced the plant growth during acclimatization (Table 2 and
Figure 3). In sand substrate, there was a significant difference between the two genotypes,
“Mejji” and “R’zwa”. In fact, the average stem growth per plantlet was 2.7 cm in “Mejji”
and 0.8 cm in “R’zwa” (Table 2). The highest stem growth (2.0 cm) in genotype “R’zwa”
was observed in peat substrate.
 Plants 2021, 10, 1062 7 of 15

Table 2. Effects of different substrate mixtures on survival rate, the number of neoformed shoots and stem growth during
ex vitro acclimatization of argan plantlets.

Survival Rate (%) Average Number of Stem Growth Stem Growth

Substrate Mixture Neoformed Shoots (cm/Plant) (cm/Week)
Peat:Sand:Forest
Soil “Mejji” “R’zwa” “Mejji” “R’zwa” “Mejji” “R’zwa” “Mejji” “R’zwa”
S1 = 1:1:1 73.3 b ± 6.6 83.3 b ± 3.3 0.2 a,b ± 0.1 0.2 b ± 0.1 1.9 a,b ± 0.2 1.3 a,b ± 0.2 0.4 a,b ± 0.0 0.3 a ± 0.0
S2 = 1:1:0 100.0 a ± 0.0 100.0 a ± 0.0 0.4 a ± 0.1 0.0 a ± 0.0 } 0.9 b ± 0.2 0.9 a ± 0.2 0.2 b ± 0.0 0.2 a ± 0.0
S3 = 1:0:0 100.0 a ± 0.0 100.0 a ± 0.0 0.0 b ± 0.0 0.0 a ± 0.0 1.3 b ± 0.2 2.0 b ± 0.3 0.3 b ± 0.0 0.5 a ± 0.0
S4 = 0:1:0 100.0 a ± 0.0 86.6 b ± 3.3 } 0.4 a ± 0.1 0.0 a ± 0.0 } 2.7 a ± 0.6 0.8 a ± 0.1 } 0.7 a ± 0.1 0.2 a ± 0.0 }
Plants 2021, 10,
Datax are
FOR PEER
means ± REVIEW
SD. Values in the same column and with different letter(s) are significantly different at p < 0.05. } indicates significance 8 of 16
between genotypes at p < 0.05. Substrate mixtures: S1, peat + sand + forest soil; S2, peat + sand; S3, peat; S4, sand.

FigureFigure
3. Acclimatization of argan
3. Acclimatization plantlets.
of argan (a–c)
plantlets. (a–c)plantlets
plantlets of
of genotypes “Mejji”(a)(a)
genotypes “Mejji” andand “R’zwa”
“R’zwa” (b,c)(b,c)
afterafter 4 weeks
4 weeks in thein the
glasshouse. (d) Plantlet growth after 10 weeks in the glasshouse. (e) Plantlet growth after 16 weeks in the glasshouse.
glasshouse. (d) Plantlet growth after 10 weeks in the glasshouse. (e) Plantlet growth after 16 weeks in the glasshouse.

The effect of the substrate mixture differed between the two genotypes. The use of
3. Discussion
peat–sand mixture or sand alone showed the formation of an average of 0.4 shoots per
Arganintree
plantlet is onewhile
“Mejji”, of thenomost
newrecalcitrant
shoots werespecies in in
observed tissue culture.
“R’zwa” Indeed,
(Table theuse
2). The adven-
titious root formation is very difficult to achieve, while it is critical for successful
of peat alone did not promote the formation of new shoots in both genotypes, whereas vegeta-
tivethe
propagation of this
peat, sand, and species
forest [12]. The
soil mixture wasdevelopment of healthy
the only substrate and vigorous
that promoted new shootroots is
essential for in
formation successful acclimatization
both genotypes toplantlets
(Table 2). The ex vitrogrown
conditions. In a previous
in peat alone study by our
exhibited vigorous
growth,
group [24],green leaves, and
adventitious well-developed
shoot rooting was andachieved
elongatedon
secondary roots.
media containing both IBA (1.5
mg L−1) and putrescine (160 mg L−1). The beneficial effects of polyamines in general and
putrescine in particular on rooting were reported in many other plant species, such as
olive [30,33], hazelnut [34], almond [35] and teak [36]. Cristofori and al. [37] showed that
putrescine, spermidine and spermine improve root formation of microshoots treated with
IBA. Many other authors (e.g., Chilley et al. [38]; Weiss and Ori [39]; Saini et al. [40]) re-
ported that products, such as jasmonic acid, strigolactones and polyamines interact either
Plants 2021, 10, 1062 8 of 15

3. Discussion
Argan tree is one of the most recalcitrant species in tissue culture. Indeed, the ad-
ventitious root formation is very difficult to achieve, while it is critical for successful
vegetative propagation of this species [12]. The development of healthy and vigorous roots
is essential for successful acclimatization to ex vitro conditions. In a previous study by
our group [24], adventitious shoot rooting was achieved on media containing both IBA
(1.5 mg L−1 ) and putrescine (160 mg L−1 ). The beneficial effects of polyamines in general
and putrescine in particular on rooting were reported in many other plant species, such
as olive [30,33], hazelnut [34], almond [35] and teak [36]. Cristofori et al. [37] showed
that putrescine, spermidine and spermine improve root formation of microshoots treated
with IBA. Many other authors (e.g., Chilley et al. [38]; Weiss and Ori [39]; Saini et al. [40])
reported that products, such as jasmonic acid, strigolactones and polyamines interact either
synergistically or antagonistically with auxins to trigger different types of events that lead
to root formation and development. On the other hand, Cristofori et al. [37] reported that
polyamines have a limited positive effect on rooting when applied without IBA. This is in
good agreement with our findings. In fact, the results of the present study showed that
using putrescine alone did not promote root induction in argan shoots. Moreover, it was
found that putrescine induces callus formation, which is probably due to its effect on cell
division [41,42]. According to Hartmann and Kester [43] and Hartmann et al. [44], root
formation can be seen from established calli or not. In the present study, callus formation
was higher in shoots cultured on RM medium supplemented with putrescine, IBA and
1.5 mg L−1 NAA. Moreover, the roots initiated from calli were fragile and easily detachable.
Our results also showed that combining IBA and NAA resulted in a higher rooting
frequency than that observed when IBA was used alone. However, increasing NAA
concentration decreased the rooting percentage. This highlights the specific requirement of
argan shoots in terms of auxin types and concentrations for efficient in vitro rooting. The
effects of NAA and IBA on root induction have been evaluated by different researchers
during both in vitro and conventional propagation of argan. Justamante et al. [18] reported
that the combination of 1 mg L−1 NAA and 1 mg L−1 IBA inhibited adventitious rooting
in seedling-derived shoots. However, in shoots produced vegetatively from microcuttings,
Lamaoui et al. [19] recommended the combination of 5 mg L−1 IBA and 1 mg L−1 NAA for
root induction, whereas Koufan et al. [17] recommended the combination of 0.5 mg L−1
NAA and 0.5 mg L−1 6-benzylaminopurine (BAP). On the other hand, Benbya et al. [45]
found that IBA is more effective than NAA in inducing adventitious roots from semi-hard
wood cuttings. These divergent results could reflect different hormonal requirements
among argan genotypes and explant types (juvenile, rejuvenated and adult material).
The effects of AgNO3 on in vitro rhizogenesis were also investigated. The findings of
the present study showed that AgNO3 promotes root formation and the development of
vigorous roots within 30 days of culture, with no callus formation. The optimal concentra-
tion of AgNO3 was 2 mg L−1 . This concentration improved the root system of shoots, which
results in increased plantlet survival and better growth and development. In many plant
species, such as Prosopis cineraria [46], Vanilla planifolia [47], Coffea arabica, C. canephora [48],
and Rotula aquatica [49], AgNO3 has improved in vitro rooting. Silver nitrate is known to
be an ethylene inhibitor. In fact, Ag+ ions produced in the culture medium play the role of
an ethylene receptor, thus ensuring easy assimilation of the energy available in the culture
medium, which improves cell division and growth. Venkatachalam et al. [46] reported
a similar effect of AgNO3 on rooting of the recalcitrant medicinal tree Prosopis cineraria.
Klíma et al. [50] showed that silver ions increase plasma membrane permeability for water
and small organic compounds as well as auxin efflux. Kumar et al. [51] reported in their
review that adding silver ions to culture medium regulates the ethylene activity and im-
proves organogenesis, in vitro rooting, flowering and shoot development. Nonetheless, the
mechanism of action of silver ions remains unclear. According to Kumar et al. [51], silver
ions target functionally interlinked ethylene, polyamine and calcium-mediated pathways.
Ruzicka et al. [52] indicated that ethylene supports auxin biosynthesis and modulates
Plants 2021, 10, 1062 9 of 15

its distribution in Arabidopsis seedling roots. They also demonstrated that silver ions
could block ethylene-induction in auxin changes. Bais [53] reported that AgNO3 exerts a
feedback inhibition on ethylene synthesis and stimulates polyamine biosynthesis by an
enhanced use of S-adenosylmethionine. Ethylene can also influence callus formation [54].
These combined actions are probably due to an interplay between polyamine and ethylene
biosynthesis, which regulates in vitro morphogenetic responses [55].
The findings of the present investigation showed that successful in vitro rooting and
development of vigorous roots in argan shoots was obtained when NH4 NO3 concentration
was reduced to 825 mg L−1, and the culture medium was supplemented with auxins (IBA
and NAA), putrescine and AgNO3 . This results in vigorous root development within only
10 days of culture. This reflects the specific requirements of argan microshoots for root
induction. In fact, some components of the culture medium allow more cells to be used
for root induction, which improves the rooting ability of shoots [56]. Similar results were
observed in other species. The beneficial effect of low NH4 NO3 concentration on in vitro
rooting was first demonstrated in apple [57]. Vahdati et al. [58] found that media with
doubled NH4 NO3 concentration reduced the rooting ability of Persian walnut. In Lavandula
spp., rooting was promoted by reducing the concentration of MS micronutrients to 12 or 14
and by adding NAA to the culture medium [59]. Similarly, the use of auxin-containing MS
medium with macroelements reduced to half, to one-third or to one-quarter strength was
effective for root induction in Rosa spp [60].
Plantlet acclimatization is a crucial step that determines the efficiency of the whole
micropropagation process. During acclimatization, regenerants are exposed to new growth
conditions causing biotic and abiotic stresses, such as water loss, tissue dehydration and
synthesis process reduction [61]. According to many authors (e.g., Lone et al. [31]; Ste-
fanello et al. [32]), the substrate used during acclimatization has a significant effect on
the survival and growth of the plantlets regenerated through in vitro culture. The potting
substrate is an essential factor in the success of plantlet acclimatization. In previous stud-
ies on the micropropagation of argan, different substrates were used. Koufan et al. [13]
planted argan micrografted plants in a mixture of peat and sand. Regarding argan micro-
cuttings, Nouaim et al. [12] used a substrate composed of Terragreen saturated with “Long
Ashton” nutrient solution. Lamaoui et al. [19] used a mixture of peat and sand, while Justa-
mante et al. [18] used a peat–perlite mix. The findings of the present study showed that the
argan plantlets grown in peat–sand mixture or in peat alone exhibit remarkable adaptation
to ex vitro conditions as well as good growth and development. The significant impact
of the substrate on plant survival and growth can be explained by its physicochemical
properties. In fact, the substrate must be porous and well-drained. In addition, it should
facilitate the acquisition of water and nutrients and allow gas exchange. This will promote
plant growth and survival and the development of a good root system [62].

4. Materials and Methods

4.1. Culture Conditions
The basal medium used in the present study was that of MS medium [63] containing
MS macroelements, MS microelements and MS vitamins. Moreover, modified MS media
were also used: RM, Murashige and Skoog basal medium modified by reducing NH4 NO3
concentration to 825 mg L−1 ; and M, Murashige and Skoog basal medium modified by
omitting NH4 NO3 . All media were supplemented with 30 g L−1 sucrose and 8 g L−1 agar.
The medium pH was adjusted to 5.8 before autoclaving at 120 ◦ C and 103 kPa for 20 min.
The chemicals used in this study were purchased from Sigma (St. Louis, MO, USA).

4.2. Plant Material and Experiments

4.2.1. Origin of Plant Material
Mature seeds of argan genotypes “Mejji” and “R’zwa” were harvested from trees
located in Essaouira province, Morocco. The seeds of the genotype “R’zwa” were col-
lected from a tree located approximately 7 km southeast of Essaouira city (31◦ 260 48.1700 N
Plants 2021, 10, 1062 10 of 15

9◦ 430 43.7400 W), while those of the genotype “Mejji” were collected from a tree located about
45 km east of Essaouira city (31◦ 320 49.7100 N 9◦ 220 32.4400 W). From each tree, 100 seeds
were collected.
Both the trees are characterized by high fruit yield. However, they are morphologically
Plants 2021, 10, x FOR PEER REVIEWdifferent. Genotype “Mejji” has a bigger size and produces round-shaped fruits, 11 of 16
while
genotype “R’zwa” produces ellipsoid-shaped fruits (Figure 4).

Figure 4.
Figure 4. Argan
Argan trees
trees and
and seeds.
seeds. (a)
(a) genotype
genotype “Mejji”
“Mejji” and
and (b)
(b) genotype
genotype “R’zwa”.
“R’zwa”.

The seeds were surface-sterilized by immersion in 70% ethanol for 2 min, min, followed
followed
by immersion in 30% sodium hypochlorite solution solution containing
containing a few drops of Tween-20
for 15 min. The seeds were then immersed in 0.1% mercury chloride for 10 min, followed
by 3 rinses with sterile distilled water, then then by
by immersion
immersion in in 0.2%
0.2% polyvinylpyrrolidone
polyvinylpyrrolidone
for 60
solution for 60min
min[24].
[24].To
Topromote
promotegermination,
germination, thethe seeds
seeds were
were soaked
soaked in mg
in 10 10 mg L−13
L−1 GA
GA12
for 3 for 12 hthen
h and and then cultured
cultured for 35for 35 days
days on sucrose-free
on sucrose-free 1/2MS 1/2MS
medium medium modified
modified by
by omit-
omitting
ting ammonium
ammonium nitrate
nitrate [24]. [24].
The epicotyl
The epicotyl explants
explants were
were excised from in
excised from in vitro
vitro germinated
germinated seedlings
seedlings andand cultured
cultured
on semi-solid MS medium supplemented with 2 mg L −1 BAP for 4 weeks under dark
on semi-solid MS medium supplemented with 2 mg L BAP for 4 weeks under dark con-
−1

conditions
ditions to induce
to induce organogenesis.
organogenesis. The The organogenesis
organogenesis rate rate
waswas
79.1%79.1%
(1.8 (1.8 shoot
shoot budsbuds
per
per explant)
explant) in “Mejji”
in “Mejji” and and
62.5%62.5%
(1.9 (1.9
shootshoot
budsbuds per explant)
per explant) in “R’zwa”
in “R’zwa” [24].[24]. For shoot
For shoot bud
bud proliferation,
proliferation, the the organogenic
organogenic cultures
cultures were
were transferredtotoMS
transferred MSmedium
medium supplemented
supplemented
with 1 mg L −1 BAP and 2 mg L−1 gibberellic acid (GA ) for 4 weeks under a 16 h photope-
with 1 mg L BAP and 2 mg L gibberellic acid (GA3) for 4 weeks under a 16 h photoperiod
−1 −1 3
[24]. The adventitious shoots obtained through this organogenesis pathway were used in
the present study (Figure 5).
Plants 2021, 10, 1062 11 of 15

Plants 2021, 10, x FOR PEER REVIEWriod [24].

The adventitious shoots obtained through this organogenesis pathway were12used
of 16

in the present study (Figure 5).

Figure
Figure 5.
5. Adventitious
Adventitious shoots
shoots of
of argan
argan used
used for
for rhizogenesis
rhizogenesis experiments.
experiments. (a)
(a) genotype
genotype “R’zwa”
“R’zwa” and
and (b)
(b)genotype
genotype“Mejji”.
“Mejji”.
Bars correspond to 1 cm.
Bars correspond to 1 cm.

4.2.2. In
4.2.2. In Vitro
Vitro Rhizogenesis
Healthy shoots
Healthy shoots (2–3(2–3 cm long) of argan genotypes “Mejji” and “R’zwa”, obtained
through direct
through direct organogenesis
organogenesis as previously described, described, were singled out and cultured on
PGR-free MS medium
PGR-free medium for for 22 weeks.
weeks. Subsequently,
Subsequently, the shoots were transferred to different
rooting media
rooting media(MS (MSor ormodified
modifiedMS MSmedia)
media)supplemented
supplemented with
with various
various concentrations
concentrations of
of IBA (0 or 1.5 mg L −1 ), NAA (0, 0.5, 1 or 1.5 mg L −1 ), putrescine (160−1mg L−1 ) and/or
IBA (0 or 1.5 mg L ), NAA (0, 0.5, 1 or 1.5 mg L ), putrescine (160 mg L ) and/or AgNO3
−1 −1

AgNO −1 ) (Table 1). The explants were placed in 300 mL glass jars
(2, 4 or 36 (2,
mg4L−1 or) (Table
6 mg L 1). The explants were placed in 300 mL glass jars (12 cm in height,
(12 cm
6.5 cm inindiameter)
height, 6.5containing
cm in diameter)
40 mL containing 40 mL ofand
of culture medium culture medium
sealed and sealed plas-
with transparent with
transparent ◦
at 25 C ±
tic lids. The plastic
cultures lids.
wereThekept
cultures
at 25 were
°C ± 1kept
in darkness 1 in
for darkness
7 days for 7 days
and then and then
transferred to
transferred to a 16 h photoperiod (40 µmol m −2 s−1 provided by cool white fluorescent
a 16 h photoperiod (40 μmol m s provided by cool white fluorescent tubes). This exper-
−2 −1
tubes).lasted
iment This for
experiment
100 days,lasted
and the forcultures
100 days, andtransferred
were the cultures towere
freshtransferred
medium at to fresh
4-week
medium at 4-week intervals. The percentage of shoots forming roots,
intervals. The percentage of shoots forming roots, the mean number of roots per shoot and the mean number
of roots
the per length
average shoot and the average
of roots length of
were recorded roots
every wereof
5 days recorded
culture.every 5 daysthe
Moreover, of amount
culture.
Moreover, the amount of callus formed was visually estimated as follows:
of callus formed was visually estimated as follows: 0C: no callus, C+: low amount of callus, 0C: no callus,
C++: moderate callogenesis, C+++: high amount of callus. The time required for rootThe
C+: low amount of callus, C++: moderate callogenesis, C+++: high amount of callus. in-
time required
duction for root induction
was determined wasinspecting
by visually determinedthe bycultures
visuallyevery
inspecting the
5 days. Thecultures every
experiment
5 days. The experiment was performed in three replicates, with 30 shoots per treatment,
was performed in three replicates, with 30 shoots per treatment, and was repeated twice.
and was repeated twice.
4.2.3. Ex Vitro Acclimatization
4.2.3. Ex Vitro Acclimatization
The
The rooted
rooted plantlets
plantlets obtained
obtained from from the
the best
best treatment
treatment were
were removed
removed fromfrom the
the rooting
rooting
medium, and their roots were carefully washed with distilled water
medium, and their roots were carefully washed with distilled water to remove adhered to remove adhered
agar.
agar. The plantlets were then transferred to plastic pots (one plant per pot)
The plantlets were then transferred to plastic pots (one plant per pot) containing autoclaved containing
autoclaved
mixtures ofmixtures of and
sand, peat sand,forest
peat soil
and in
forest soil inproportions
different different proportions
1:0:0, 0:1:0,1:0:0,
1:1:1,0:1:0,
and1:1:1,
1:1:0
and
(w/w/w) and placed in the growth room. To maintain high humidity, the pots werethe
1:1:0 (w/w/w) and placed in the growth room. To maintain high humidity, pots
covered
were covered with transparent polyethylene bags. After 2 weeks, the plantlets
with transparent polyethylene bags. After 2 weeks, the plantlets were transferred to the were trans-
ferred to the They
glasshouse. glasshouse. They were maintained
were maintained at 25 ± 2 ◦ C at under
25 ± 2 light
°C under light conditions
conditions and regularly and
regularly irrigated
irrigated with with Hoagland
Hoagland and Arnon’s andnutrient
Arnon’ssolution
nutrient[64].
solution [64]. Meanwhile,
Meanwhile, the pol-
the polyethylene
yethylene
bags werebags were gradually
gradually removed.removed. Finally,
Finally, the the plantlets
plantlets were transferred
were transferred to larger toplastic
larger
plastic pots. After 4 weeks in the glasshouse, the survival rate, the number
pots. After 4 weeks in the glasshouse, the survival rate, the number of neoformed shoots, of neoformed
shoots,
and theand thegrowth
plant plant growth (i.e.,
(i.e., the the initial
initial size
size of theofplantlets
the plantlets
and and
the the
sizesize after
after 28 days
28 days of
of transplantation) were recorded. Three replicates of 5 pots each were used
transplantation) were recorded. Three replicates of 5 pots each were used per substrate, per substrate,
and
and the experiment was repeated twice.
Plants 2021, 10, 1062 12 of 15

4.3. Data Analysis

Data are expressed as means and standard deviations (SD). All experiments were set
up in a completely randomized block design. One-way analysis of variance (ANOVA I)
was performed to test for significant differences among the different treatments. The
least significant difference test (LSD) was used for post hoc mean separation at the 5%
significance level. Tests for normality and homogeneity of variance were performed before
ANOVA. Before analysis, all percentage data were arcsine transformed.

5. Conclusions
We described an efficient protocol for in vitro rooting and acclimatization of micro-
propagated argan shoots. The optimal medium for shoot rooting was RM medium (MS
medium modified by reducing NH4 NO3 concentration to 825 mg L−1 ) supplemented with
160 mg L−1 putrescine, 2 mg L−1 AgNO3 , 1.5 mg L−1 IBA and 0.5 mg L−1 NAA. In this
medium, rooting started after only five days of culture. Moreover, the development of a
healthy and vigorous root system was observed, with no or low callus formation. In fact,
adding putrescine, NAA, IBA and AgNO3 promoted root formation, inhibited callogenesis
and reduced the time required to produced rooted plantlets ready for acclimatization.
AgNO3 had an inhibitory effect on callus formation and improved the quality of the root
system. The addition of NAA at low concentration increased the percentage of rooting and
the number and length of roots. Regarding acclimatization, a high survival rate of 100%
and normal growth and development of plants were observed in the peat–sand mixture
or when peat was used alone. The reported results will be used for rapid and large-scale
propagation of this endangered plant species and to rehabilitate the argan ecosystem.
Further studies are currently undertaken to test the developed protocol’s suitability on
other superior argan genotypes characterized by high fruit yield and high oil content.

Author Contributions: Conceptualization, experiment design and methodology, R.A., G.D., F.G. and
M.I. (Mohammed Ibriz); Plant material collection and preparation, I.A., M.I. (Maha Ibrahimi) and
A.B.; experiments and data analysis, I.A., M.A.M., R.M. and F.G.; data curation, R.A., G.D. and D.I.;
writing—original draft preparation, I.A., A.B., M.I. (Maha Ibrahimi), and F.G.; writing—review and
editing, R.A., G.D., M.A.M., R.M. and D.I.; supervision, R.A. and M.I. (Mohammed Ibriz); funding
acquisition and project administration R.A., G.D. and D.I. All authors agreed to be accountable for all
aspects of the work. All authors have read and agreed to the published version of the manuscript.
Funding: The present investigation was fully funded by the National Institute of Agronomic Research
of Morocco (INRA-Morocco).
Conflicts of Interest: The authors declare no conflict of interest.

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